Proteins are exceedingly versatile biomacromolecules. One class of proteins known as enzymes functions as perhaps nature's most perfect catalysts for effecting complex organic syntheses. Because enzymes function so efficiently at low concentrations causing a high conversion of reactants to products under extremely mild conditions of temperature and pH and do so with an incomparable degree of specificity (in terms of functional groups affected), they have long been sought after by organic chemists as catalysts to conduct chemical reactions. Medicinal and pharmaceutical chemists have further been intrigued by the ability of enzymes to synthesize just one of the possible optical isomers of a compound wherein the simple spatial arrangement of four different substituents on a particular carbon atom gives rise to unique pharmacological behavior. Interest in enzymes for synthetic purposes has also heightened recently by advances in molecular genetics that have allowed for the preparation of specific enzymes in a highly purified state, on a relatively large scale, and at substantially reduced cost.
Insolubilization of enzymes without losing catalytic activity has been an objective of many investigators because of the very practical advantages that catalyst systems can be easily removed from reaction mixtures by simple filtration and, at least in principle, can be reused many times. Other factors which contribute to cost savings of insolubilized enzyme catalysts over soluble enzymes and are normally accrued in the insolubilization process include enhanced stability over a wider range of temperature and pH.
The most common technique of employing immobilized or insolubilized enzymes to catalyze a chemical reaction is to simply add the insoluble particulates to a solution containing dissolved reactant. The insoluble catalyst is stirred, often at high rates, to facilitate reaction by more efficiently mixing catalyst and reactants. This stirring often causes mechanical breakup of the insoluble enzyme particulates which can give rise to leaching, enzyme deactivation, and increased separation costs, as smaller particulates are generally more difficult to remove from the reaction mixture. Also, if pH control is maintained by simultaneous addition of strong acid or base, the insolubilized enzyme will almost certainly be exposed to momentary pH extremes before the titrant can mix into the reaction mixture. In aqueous solution amino acid residues such as lysine, aspartic acid, glutamic acid, and others ultimately define protein structure because they are frequently charged and present on the periphery of the protein's tertiary structure. Sudden changes in pH can either protonate or deprotonate groups on these amino acid residues.
The above-mentioned deficiencies of batchwise employment of an insoluble enzyme as a heterogeneous component in a reaction mixture have led to the development of various flow reactors employing packed columns of insoluble enzyme particulates. While these reaction systems minimize mechanical instability, problems of modest capacity and pH instability remain. Catalyst capacity generally corresponds directly with column length, and with longer columns undesirably high pressures are required in order to achieve reasonably rapid throughput of solution. Also, pH control is generally completely lacking within the column where deleterious effects on the enzyme can occur.
Liquid cartridge filters have been developed over the years that operate at relatively high flow rates, e.g., liters per minute, and at relatively low pressures. In tangential flow or radial membrane cartridge filters, the filtering element is presented in a plane parallel to the liquid stream flow, and two effluents or permeates are produced, one filtered or processed by passing through the filtering element and another not. While these filter arrangements operate at low pressures and the unprocessed permeate can in theory be recycled, these systems are intrinsically more complicated and slower to completely process a liquid stream because of relatively low flow through the element; also, if modified to insolubilize enzymes, complete conversion of reactant would be required in one pass through the element.
In "dead end" filters the filtering element is presented perpendicularly to the direction of flow of the liquid stream. All the liquid stream is required to pass through the element and only one permeate is produced. Considered as a reactor in which reaction is occurring on or within the filtering element, the dead end cartridge filter would be analogous to a very wide, but short column. Because the actual length of the catalyst layer is relatively short, changes in pH caused by the enzymatic reaction should not be large at relatively high solution throughput. At high flow rates single pass conversion may be relatively low but by repeatedly cycling the effluent high conversion could be achieved, with pH being adjusted between passes. Attributes of recirculation to minimize diffusional effects, at least in analytical assays involving immobilized enzymes, are discussed by J. R. Ford, et al., Biotechnol. & Bioeng. Symp. No. 3, 1972, 267-284.
T. J. Harrington, et al., Enzyme Microb. Technol., 1992, 14, 813-818 describe a ceramic microfilter employed in a recycling dead end configuration in which an alumina microfilter (0.45 micrometer nominal filter rating) has been activated with aminosilane/glutaraldehyde to accommodate enzyme attachment.
Efforts to improve capacity and lower operating pressures of dead end filter cartridges have utilized the high surface areas of particulates that can bind with and insolubilize enzymes. These efforts have utilized insolubilized particulates contained within a filtering layer itself and single pass operations. Canadian Patent No. 1,179,283 discloses a cartridge filter in which silica gel is embedded within a poly(vinyl chloride) membrane. An enzyme, insolubilized by adsorption onto the silica gel, was employed to isomerize sucrose to isomaltose. Similarly, U.S. Pat. No. 4,857,461 discloses a single pass, continuous process in which a dead end cartridge filter was utilized which contained, again within a web matrix, a crosslinked cellulose cation exchange resin possessing sulfonic acid groups. Enzymes were adsorbed via ion exchange, post crosslinked with glutaraldehyde, and utilized to isomerize sucrose in 97% yield with single pass flow rates up to about 12 mL/minute (flux rate=0.005 cm/minute). "Pressure driven enzyme membrane reactors" with flux rates as high as 0.20 cm/minute are more fully described by Schmidt-Kastner, et al., Biochem. Eng. [Intl. Congr.], 1986 (publ. 1987), 111- 131 in which enzyme supported silica particulates are embedded within the porous structure of a membrane.